The long-term goals of this project are to reveal mechanisms by which adipocytes function to regulate energy balance and metabolism in humans. Adipocytes secrete a variety of physiologically important proteins including the adipokines (adipose-derived cytokines) leptin and adiponectin. The assembly and secretion of these and other adipokines is initiated in the endoplasmic reticulum (E.R.) where various chaperones and redox-sensitive enzymes are involved in protein folding and disulfide bond formation. We recently discovered a novel membrane protein of the E.R. that is a member of the thioredoxin/peroxiredoxin family. We named it adiporedoxin (Arx) because it is expressed to a significant degree only in white and brown in adipocytes as well as in testes, and its function, like that of related proteins, is to assist in disulfide bond formation and protein folding. Our preliminary data show that by altering the expression of adiporedoxin, we can corresponding alter the levels of secreted adipsin (a complement-related adipokine) and adiponectin, an important adipokine whose levels correlate positively with insulin sensitivity. Moreover, over and under expression of adiporedoxin in cultured mouse adipocytes causes corresponding changes in the amount of the insulin receptor. What these three proteins have in common, aside for important physiological functions, is the need for multiple disulfide bonds in their tertiary and quaternary structure. Our preliminary data support the hypothesis that adiporedoxin may be a major regulator of adipocyte protein assembly and secretion. We will test this hypothesis in Aim1 where mechanistic properties of Arx related to protein assembly and secretion will be investigated in co-transfection experiments with adipokines. In Aim2 we will use adipocytes and various Arx constructs to determine how this protein functions in its physiological milieu in vitro We will interrogate the entire proteome, the secretome and the plasma membrane proteome of Arx over and under expressing fat cells. In Aim3 will determine the effects of endoplasmic stress and obesity on Arx function with regard to adipokines and other possible substrates.. Taken together, the proposed experimentation will reveal fundamental aspect of eukaryotic biology, the function of the endoplasmic reticulum in protein folding and disulfide bond assembly. This very important area of quality control for proteins secreted from adipocytes has been minimally investigated, and the topic has important consequences for human health and disease. If adipokine production is disrupted by changes in adiporedoxin expression, it is highly likely that long-term negative consequences for the organism will occur such as obesity and insulin resistance. By understanding the mechanism of Arx action, these consequences can be addressed by appropriate therapeutic strategies. PUBLIC HEALTH RELEVANCE: Adipocytes or fat cells produce a variety of hormone-like substance and release them into the blood, after which they act on tissues such as muscle and brain to regulate body weight and have anti-diabetic properties. It is the goal of this proposal to describe the molecular details by which these hormone-like substances are made and assembled in the fat cell because these cellular events determine circulating hormone levels, and in turn, the response of other tissues, which may include disease-causing abnormalities if their assembly is compromised.